Rocky Mountain spotted fever, the most prevalent and important of the rickettsioses in the United States, is an acute tick-borne febrile illness caused by Rickettsia rickettsii. The organism infects and proliferates predominantly within vascular endothelial cells, which respond by activating a series of defense mechanisms, possibly via distinct signal transduction pathways. We have demonstrated that R. rickettsii infection of endothelial cells results in the activation of nuclear factor-kappaB (NF-kB a transcription factor which controls the expression of an array of genes involved in bacterial infections, immune response, and apoptosis. We have also shown that anti-apoptotic functions of NF-kB protect the host cell from apoptotic death during R. rickettsii infection. The objective of this application is to further our understanding of signaling mechanisms underlying Rickettsia-induced transcriptional activation and investigate their participation in the host cell response to infection. Since the seminal event in the activation of NF-kB is the degradation of IkB (inhibitors of NF-kB) proteins by IkB kinase (IKK) complex, Aim I will characterize the activation of IKK and phosphorylation/degradation of IkB proteins during infection. We will determine the kinetics of activation of catalytic subunits, IKK-a and IKK-b, by an immunoprecipitation (IP) kinase assay. The role of the regulatory subunit, IKK-g will be evaluated using a specific, cell permeable peptide that blocks its association with the IKK complex. Aim 2 will characterize the activation of mitogen activated protein (MAP) kinases and investigate their involvement in rickettsial invasion of endothelial cells and activation of NF-kB Modulation of MAP kinase cascades, ERK1/2 and p38, will be examined by immunoblotting using phosphorylation state specific antibodies and activity assays by IP western analysis. Aim 3 will define the regulation of chemokine induction in response to infection and explore its dependence on the MAP kinase and NF-kB pathways. Using molecular biology and microscopy techniques and strains of Rickettsia with varying pathogenicity, we will investigate the correlation between infection, activation of IKK/NF-kB and MAP kinases, and induction of chemokine response. These studies will offer important perspectives in understanding rickettsial pathogenesis and provide valuable insight into the complex interplay of signaling events, which occur as part of the host-parasite relationship.